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At least 19 recordsLinked to original sources
PubMed2004

Possible involvement of the 5'-flanking region and the 5'UTR of plastid accD gene in NEP-dependent transcription.

In many developmentally and functionally important higher plant plastid genes, expression depends on a specific nuclear-encoded RNA polymerase (NEP). Molecular mechanisms for NEP-mediated gene expression are poorly understood. We have improved a transient expression assay based on biolistics and the dual-luciferase reporter technique, which facilitated investigations into the regulation of plastid genes in vivo. We scrutinized the 5'-flanking region and the 5'-untranslated region (5'UTR) of accD, a plastid gene encoding a subunit of the prokaryotic-type acetyl-CoA carboxylase which is transcribed exclusively by NEP. The results indicated that two AT-rich sequences, one of them containing two overlapping YRTA-like motifs, were essential for accD expression in vivo. The results also revealed that the length of the 5'UTR rather than a particular sequence element was a determinant for the level of accD expression. Because transcripts accumulated in proportion to reporter enzyme activity and protein levels, and transcript degradation rates were independent of the nature of the 5'UTR, it was unlikely that the 5'UTR acts as a translational enhancer or a stabilizer of the transcripts. Therefore, the length of 5'UTR might be a factor contributing to the efficiency of NEP-dependent transcription in plastids.

5' Flanking Region↗
PubMed1990

Characterization of the transcription-initiation site and of the promoter region within the 5' flanking region of the human aldolase C gene.

Several aldolase C clones from a human genomic library have been identified using a mouse aldolase C cDNA as a hybridization probe. The most complete fragment of the clones identified is 14 kb long and contains the complete aldolase C gene. The nucleotide sequence analysis of more than 5 kb includes the intron/exon organization structure of the gene and the 3' and 5' flanking regions. Although no human cDNA is yet available, a canonical polyadenylation signal at the 3' end of the gene indicates the proximity of the poly(A) addition site. We have analyzed the 5' noncoding region by S1 mapping and primer-extension experiments. The transcription-initiation sites for the human aldolase C gene in brain tissue was located about 1300 bp upstream from the methionine initiation codon. Preliminary functional assays of the promoter by transfection into rat glioma cells have indicated that promoter elements lie between positions -161 and -416 from the start point of transcription.

Amino Acid Sequence↗
PubMed2000

Characterization of an androgen-specific response region within the 5' flanking region of the murine epididymal retinoic acid binding protein gene.

The epididymis provides the optimal milieu for sperm maturation and storage. Epididymal secretory proteins are believed to be involved in that process. Androgens are the major endocrine and paracrine regulatory signals that regulate gene expression in the epididymis. We have previously identified an androgen-dependent retinoic acid-binding protein (mE-RABP) that is secreted into the luminal fluid from the mouse mid/distal caput epididymidis. The mE-RABP protein belongs to the lipocalin superfamily and may be involved in the trafficking of retinoic acid within the epididymis. We have recently demonstrated that 5 kilobases of the 5' flanking region of the mE-RABP gene contained all the information for the hormonal regulation and the tissue-, region-, and cell-specific expression of the mE-RABP gene. In this study, we have identified a complex androgen-specific response region (ARR) within the first 600 base pairs of the mE-RABP gene promoter. Androgen (DHT) but not glucocorticoid (DEX) activates the ARR in HeLa and PC-3 cells. Two androgen receptor binding sites have been located at positions -445/-459 and -102/-88 and were named ARBS-1 and ARBS-0, respectively. Point mutations of ARBS-0 resulted in a slight decrease of the androgen response. However, mutations of ARBS-1 led to a total loss of the androgen responsiveness, suggesting that it was a major cis-acting element. When ARBS-1 is isolated from its promoter context, it serves as a weak androgen-responsive element that was activated by both androgens and glucocorticoids. Also, the -543/-88 DNA promoter fragment behaved as a poor androgen-responsive region, suggesting that regulatory elements located within the proximal mE-RABP promoter were required for a full androgen response. In conclusion, the mE-RABP ARR is a good model for the study of molecular mechanisms that lead to an androgen-specific responsiveness in vivo.

Androgens↗
PubMed1995

DNA sequence of the porcine alpha-lactalbumin 5' flanking region and single-base polymorphisms within this region.

The 5' flanking region of the alpha-lactalbumin (alpha-LA) gene was sequenced for the Duroc, Yorkshire and Meishan breeds of swine to identify potential sequence variants within this regulatory region of the porcine alpha-LA gene. The sequenced region of the gene encompasses 391bp5' of the translation start site to 11bp3' of the translation start site. Within this sequence of the porcine alpha-LA gene two single-base pair differences were detected. One variant occurs at position -178 and the other at position -235 from the translation start site. Each of the variations can be detected by a restriction fragment length polymorphism within a polymerase chain reaction amplified product. The polymorphisms at the -178 and -235 positions appear to be genetically linked in the animals that have been analysed.

Animals↗
PubMed2001

Analysis of the coding and the 5' flanking regions of the alpha-synuclein gene in patients with Parkinson's disease.

Missense mutations of the alpha-synuclein gene have been reported to explain a few kindreds with autosomal dominant Parkinson's disease (PD). In order to identify mutations in our PD patients, we have screened the coding region and 5'flanking region of the gene. DNA samples from 50 patients with familial PD were screened via single-strand conformation polymorphism (SSCP) for mutations in the alpha-synuclein gene. The 5' flanking region was examined in 117 additional PD patients (27 patients with unclear family history for PD, and 90 patients without family history) and in 169 control subjects. We found one change (G199A) in exon 4 in one family with a pattern of autosomal dominant PD. However, this mutation did not result in an amino acid substitution (valine) and did not segregate completely with PD. The analysis of the 5' flanking region also showed a new polymorphism, a nucleotide insertion (- 164insA) linked to a nucleotide substitution (C-116G), in patients and in controls. The -164insA/C-116G allele was present in 52.3% of the patients and in 47.6% of the controls. We did not find significant differences regarding the allelic and genotype frequencies between PD and control groups. These results suggest that mutations in the alpha-synuclein gene are a very rare cause of familial PD and that the novel -164insA/C-116G polymorphism in the 5' flanking region does not confer susceptibility to develop PD.

Aged↗
PubMed2000

Structural study of the porcine Na+/H+ exchanger NHE1 gene and its 5'-flanking region.

The Na+/H+ exchanger is an ubiquitous mammalian plasma membrane protein that is important for the regulation of intracellular pH and cell volume. In order to provide some insight into the molecular basis of NHE1 expression we have isolated and characterized genomic DNA clones containing the coding region and 5'-flanking region of the porcine NHE1 gene. The gene spans more than 30 kb in length and consists of twelve exons that are flanked by typical splice donor and acceptor sequences at the exon-intron boundaries. The positions of the splicing sites are conserved in relation to the human NHE1 gene. The 5' distal transcription initiation site, identified by primer extension analysis, is positioned 766 bp upstream of the translation initiation codon and 36 bp downstream of a TATA box. A 5'-flanking region of 1.62 kb in length contains a number of potential regulatory elements, and exhibits several features that distinguish the pig gene from those of rabbit, mouse and human. The NHE1 gene is located in a CpG island. The promoter sequence of 500 bp is compared with that for NHE1 genes from different species. The homology between the porcine and the human, rabbit and mouse genes is 78, 76 and 75%, respectively. Several consensus elements for transcription factors, including AP-1, C/EBP, and Sp1 are phylogenetically conserved between pig and human, while AP3 and PEA3 are found only in pig. Some conserved elements are found in the pig in multiple copies. These results suggest broadly similar regulatory mechanisms for NHE1 transcription among the different mammalian species but show some species or tissue-specific differences.

5' Untranslated Regions↗
PubMed1986

Investigation of the polymorphic region in the 5' flanking region of the insulin gene in patients with Alzheimer's disease.

A potential relationship between Alzheimer's Disease (AD) and insulin gene expression was suggested by the observation that patients with AD have altered levels of fasting blood sugar and insulin. Since polymorphisms in the region 5' to the insulin gene have been associated with blood glucose levels, we have studied this polymorphism in AD patients. Subjects were 19 nondiabetic AD patients with symptoms of aphasia and apraxia and a family history of AD; and 20 age and sex-matched nondiabetic controls without family history of AD. The 5' polymorphic region of the insulin gene was analyzed by restriction enzyme digestion of DNA extracted from whole venous blood. We did not observe a correlation between the size of the 5' polymorphic region and AD.

Alzheimer Disease↗
PubMed2005

High level of endothelial cell-specific gene expression by a combination of the 5' flanking region and the 5' half of the first intron of the VE-cadherin gene.

To develop a tool to obtain a high level of gene expression specifically in endothelial cells (ECs), we assessed enhancer activity of fragments in the first intron of the VE-cadherin gene using 3 different experimental systems: luciferase assay in the F2 EC line, green fluorescent protein (GFP) expression in ECs generated in embryonic stem (ES) cell differentiation culture, and GFP expression in transgenic mice. Although the 2.5-kbp (kilobase pair) 5' flanking sequence of the VE-cadherin gene is EC specific, adding 4 kbp of the 5' half of the first intron affected an enhancement of the gene expression level in all 3 assay systems. No other fragments tested in this study could confer such effects. Compared with other gene expression units, the unit described in this study would be the most optimum one available to date for EC-specific gene expression. Because this unit can express genes in VE-cadherin(+) progenitors of hematopoietic cells but not in fully committed hematopoietic cells, it will be useful to manipulate specifically the uncommitted progenitor stage during hematopoietic cell differentiation.

Animals↗
PubMed2001

Structural characterization and comparison of promoter activity of mouse and bovine interphotoreceptor retinoid-binding protein (IRBP) gene 5' flanking regions in WERI, Y79, chick retina cells, and transgenic mice.

PURPOSE: To determine the sequences of the mouse and bovine interphotoreceptor retinoid-binding protein (IRBP) 5' flanking regions and whether these 5' flanking regions contain functional IRBP promoter activity in multiple cell types using both quantitative and statistical analyses. METHODS: We sequenced the bovine and mouse 5' flanking regions of the IRBP gene and compared these sequences to the human gene sequence. To test for functional activity of this region, we used the same DNA construct, p1783, in four different cell types. Mobility shift, DNase footprints, and southwestern blots were used to determine where nuclear protein complexes bind the IRBP 5' flanking region. RESULTS: The 5' flanking regions of the bovine, human, and mouse IRBP genes exhibit sequence similarity in regions immediately adjacent to the start of transcription (roughly 350 bases in length) and also over a 220 base sequence about 1.25 to 1.50 kb upstream of the transcription start site. Two different statistical approaches showed that the IRBP 5' flanking region possesses promoter activity in four different cell types. By using mobility shift, DNase I-protection experiments, and southwestern blotting, a region of about 45 bases at position -300 was identified that specifically binds a protein from the nuclei of bovine retina and Y79 cells. CONCLUSIONS: Specific DNA binding events are an essential part of IRBP promoter activity. The conservation of sequences far upstream of the transcription start suggest that unknown physiological processes remain to be understood in IRBP transcriptional regulation.

5' Flanking Region↗
PubMed1994

Study of 5'-flanking region of human Cu/Zn superoxide dismutase.

The 5'-flanking region of human Cu/Zn superoxide dismutase (SOD1) was cloned from human genomic library for the study of regulation of human SOD1 gene. We determined 3678 nucleotide sequences of 5'-flanking region of human SOD1. The putative binding sites of transcriptional factors such as NF1, Sp1, AP1, AP2, GRE, HSE and NF kappa B were found. The upstream region of this gene was analyzed by deletion and measuring the linked chloramphenicol acetyltransferase (CAT) activities. Several deletion analyses of promoter activity indicated that there were positive and negative regulatory regions. The region from -1325 bp to -1040 bp was found to have a heat shock response element.

Base Sequence↗
PubMed1994

5'-flanking region surrounding a human cytosolic phospholipase A2 gene.

The 5'-flanking region and the first four exons of a gene encoding human cytosolic phospholipase A2 (cPLA2) were isolated from a human lambda EMBL3 genomic library and sequenced. The 5'-flanking region is characterized by CA repeats, one of microsatellites. The analysis of the 5'-flanking region with transcription factor database suggests the existence of the transcription factor binding sites such as NF-kappa B, NF-IL6, AP-1, AP-2, and PEA3. These factors are well known to be induced or activated by the reagents (tumor necrosis factor alpha, interleukin-1, epidermal growth factor, and phorbol myristate acetate) reported as the inducers of a cPLA2 gene.

Base Sequence↗
PubMed2005

Polymorphisms of 5' flanking region of chicken prolactin gene.

The polymorphisms of 5' flanking region of chicken prolactin (cPRL) gene were examined in several populations of Chinese native Yuehuang, Taihe Silkie and imported White Leghorn Layer chickens. The 5' flanking regions (2638 bp) from Yuehuang, Taihe Silkie and White Leghorn chickens were subjected to sequencing analysis. Four single nucleotide polymorphisms (SNPs) were identified at position -2425(C/T), -2215(T/C), -2063(G/A) and -1967(A/G). A 24-bp indel (insertion or deletion) and a polyA length polymorphism were also identified. For the 24-bp indel locus, three genotypes (AA, AB and BB) were found in Yuehuang chickens, while only two genotypes were detected in Taihe Silkie (AB and BB) and Leghorn chickens (AA and BB). The genotype frequencies of AA, AB and BB were significantly different among the three breeds. For the polyA locus, although three genotypes (CC, CD and DD) were found, only one genotype (CC) was detected in White Leghorn chickens, while two or three genotypes were observed in Chinese native chickens. We used real-time quantitative PCR and radioimmunology assay to investigate the potential association of the 24-bp indel locus with cPRL mRNA expression, plasma cPRL and brooding behaviors, and observed that chickens with genotype AB, which are of the highest incidence of broodiness, had the highest cPRL mRNA levels, providing the possibility that this polymorphic site might be related to the broodiness in chickens via modulating the transcriptional level of cPRL gene. The dissociation among cPRL gene transcription, mRNA storage and hormone release was also observed.

5' Flanking Region↗
PubMed2005

Sequence analysis of the 5'-flanking regions of human dihydropyrimidine dehydrogenase gene: identification of a new polymorphism related with effects of 5-fluorouracil.

Dihydropyrimidine dehydrogenase (DPD), known as a rate-limiting metabolic enzyme in the catabolism of 5-fluorouracil (5-FU), degrades more than about 80% of the administered 5-FU in human liver. Since it was reported that the anticancer effects of 5-FU were observed in cancer patients with lower DPD activities, many attempts have been conducted to anticipate the expected anticancer effects of 5-FU based on expression of intracanceral DPD. It have been reported that 39 different mutations and polymorphisms in the coding regions of DPD genes have been identified; however, there is no report on polymorphisms in the 5'-flanking region of DPD genes. We investigated polymorphisms in the 5'-flanking regions (3,058 bp), which are considered to control expression of DPD genes, in genomic DNA extracted from 37 kinds of human cancer cells. As the results, out of 37 cancer cells subjected to analysis, DLD- 7 cells had C insertion and 7 strains G deletion, which were hetelozygote. No significant relationship was identified between the DPD activity and the expression levels of DPD mRNA in examined 10 kinds of human cancer cells. However, in DLD-1 cells, which have C-insertion polymorphism in 5'-flanking region of DPD gene, the DPD activity was below detection limit (< or = 0.5 pmol/min/mg protein). Furthermore, 50% of cytosine residue on the CpG site generated by the C insertion was methylated at the 5 position. In this study, we have identified novel polymorphism possibly related the cytotoxicity of 5-FU in the 5'-flanking region of DPD gene. It is suggested that newly identified polymorphism of DPD gene might affect transcription of DPD, thereby providing influence on the clinical outcome of cancer patients treated with 5-FU.

Antimetabolites, Antineoplastic↗
PubMed2005

Transcription of macrophage IGF-I exon 1 is positively regulated by the 5'-untranslated region and negatively regulated by the 5'-flanking region.

Idiopathic pulmonary fibrosis (IPF) is an insidious lung disease with no known cure or effective therapy. Macrophage-derived insulin-like growth factor-I (IGF-I) is thought to play a role in the pathogenesis of IPF; however, little is known about the control of IGF-I expression in macrophages. In this report we investigated the cis-regulatory elements that control basal expression using luciferase reporter constructs in RAW 264.7 macrophages. We show that the +95 to +329 region contains elements necessary to direct maximal promoter activity, whereas the +251 to +329 region contains the minimal promoter. Mapping transcriptional start sites for endogenous IGF-I in primary macrophages revealed that the major transcriptional start site is centered at +150, whereas the most 3'-transcriptional start site is centered at +255. Nuclear proteins from primary and RAW 264.7 macrophages bind specifically to the region required for maximal promoter activity (+134 to +173) and to the region required for minimal promoter activity (+267 to +299). Antibody supershift assays indicate that Sp3 bound to the +267 to +299 region. Moreover, mutation of the putative binding site reduced Sp3 binding in EMSAs and increased promoter activity in luciferase reporter gene assays. We also found that the regions from -1711 to -855 and -855 to -337 contain putative macrophage-specific suppressor elements that do not function in HeLa or COS-7 epithelial cell lines. These data support the view that macrophage IGF-I expression is positively regulated by elements located in the 5'-untranslated region and negatively regulated by elements in the 5'-flanking region of the IGF-I gene.

5' Untranslated Regions↗
PubMed1988

An upstream enhancer and a negative element in the 5' flanking region of the human urokinase plasminogen activator gene.

The 5' flanking region of the human urokinase (uPA) gene has been fused to the reporter chloramphenicol acetyl transferase (CAT) gene and its activity assayed by transfection in two human cell lines. Progressive deletions of the uPA regulatory region from the 5' end maintain a high level of expression provided at least 1870 (in A1251 cells) or 1963 (in HFS10 cells) nucleotides of the 5' flanking region are retained. A DNA fragment from -2350 to -1824 has enhancer properties, stimulating transcription of an enhancerless SV40 early promoter independently of orientation and distance. Internal deletions that still retain the enhancer element reveal the presence of negative cis-acting sequences between -1824 and -1572. Their removal, in fact, increases uPA transcriptional activity. Differences of expression of the uPA-CAT fusion genes in the two cell lines are also observed, indicating the presence of cell-specific cis-acting sequences.

Amino Acid Sequence↗
PubMed1998

Polymorphism in the 5' flanking region of the human somatostatin receptor subtype 5.

The human somatostatin receptor subtype 5 (hSSTR5) gene has previously been cloned and localized to chromosome 16 p13.3. This region is evolutionarily conserved in all vertebrate genomes from the puffer fish (Fugu rubripes) to human, and also contains loci for genes associated with two common multisystemic disorders, adult polycystic kidney disease (PKD1) and tuberous sclerosis (TSC2). Analysis of the 5' flanking region of the hSSTR5 gene has revealed consensus sequences for a number of transcription factors as well as Alu-like repeat elements. In the present study, genomic DNA from 53 unrelated individuals was analysed by PCR and Southern blots probed with radiolabeled fragments generated from different segments of the hSSTR5 gene. We have identified two restriction fragment length polymorphisms (RFLP) with high heterozygosity values at the 5' flanking region of the hSSTR5 gene. These RFLP markers will be useful for determining the allelic loss of genetic material from this region. The observed polymorphism in the promoter region may affect the function of the hSSTR5 gene.

Adult↗
PubMed1989

Genomic footprinting of a yeast tRNA gene reveals stable complexes over the 5'-flanking region.

We have shown by genomic footprinting that the 5'-flanking region of the Saccharomyces cerevisiae tRNASUP53 gene is protected from DNase I digestion. The protected region has a 5' boundary at -40 (relative to the transcription initiation site) and extends into the coding region of the gene, with a 3' boundary at approximately +15. Although the DNase I protection over this region was much greater than at the A- and B-box internal promoters, point mutations within the A or B box that reduced transcription in vitro eliminated the upstream DNase I protection. This implies that formation of a stable complex over the 5'-flanking region is dependent on interaction of the gene with transcription factor IIIC but that stability of the complex may not require continued interaction with this factor. The DNase I protection under varied growth conditions further suggested that the upstream complex is composed of two or more components. The region over the transcription initiation site (approximately +15 to -10) was less protected in stationary-phase cultures, whereas the more upstream region (approximately -10 to -40) was protected in both exponential- and stationary-phase cultures.

Base Sequence↗
PubMed2004

[Primary functional analysis of CK13 gene 5' flanking region].

OBJECTIVE: To investigate the mechanism of cytokeratin 13 (CK13) gene expression control and the effects of different motifs of CK13 gene 5' flanking region on its transcriptional activity. METHODS: The molecular clone technique and reporter gene analysis were used to assay the effects of different motifs of 513 bp of CK13 gene 5' flanking region on its transcriptional activity. The pCAT enhancer vectors with different motifs of CK13 gene 5' flanking region were constructed and transferred to HeLa cells with the help of lipofectin. The instant CAT expression of different clones was detected and the effects of different motifs of the CK13 gene 5' flanking region on its transcriptional activity were evaluated. RESULTS: 119 bp from -nt.325 to -nt.207 upstream of the first ATG of CK13 gene 5' flanking region included a silent element. 113 bp region from -nt.206 to -nt.94 included an enhanced element. CONCLUSION: 513 bp of CK13 gene 5' flanking region includes a silent element and an enhanced element. Further locating these cis elements and detecting the related trans reaction factors may unveil some important clues to the details of the mechanisms for the CK13 gene expression and tissue-specific expression.

5' Flanking Region↗